Because of a severe shortage of human organ donors, most candidates for a liver transplant never receive one. Currently, approximately 4000 liver transplants are performed annually. Over 11,000 high priority candidates are waiting for a transplant. It has been estimated that as many as 50,000 could benefit from a liver transplant each year, if the organs were available. Pig liver xenografts could resolve the shortage, but the problems of severe rejection and abnormal function preclude their use. This proposal will develop initial technology to produce pig liver xenografts repopulated with human hepatocytes. The livers would then produce the appropriate human proteins. The program will produce genetically engineered pigs using a new efficient method for transfecting oocytes, developed by Gala Design LLC. Other methods used in this proposal include cellular transplants, molecular biology and immunohistochemistry techniques. The technology would provide liver grafts for those without human donors. When combined with surrogate tolerogenesis, developed elsewhere, the recipients would have a significantly decreased need for immune suppression to prevent rejection. In addition to transplantation, the hybrid grafts would be useful for toxicology studies and as a model for the study of viral hepatitis. PROPOSED COMMERCIAL APPLICATION: Hybrid pig livers repopulated with human hepatocytes would provide functional liver xenografts for recipients without human donors. Chimeric pigs would be additionally valuable for the study of human diseases such as viral hepatitis and as a toxicology system for assessing injury to human hepatocytes. The system could be used as a cost effective bioreactor to expand human hepatocytes for cell transplants.